Oridonin Attenuates Burkholderia cenocepacia Virulence by Suppressing Quorum-Sensing Signaling

ABSTRACT Burkholderia cenocepacia is a human opportunistic pathogen that mostly employs two types of quorum-sensing (QS) systems to regulate its various biological functions and pathogenicity: the cis-2-dodecenoic acid (BDSF) system and the N-acyl homoserine lactone (AHL) system. In this study, we reported that oridonin, which was screened from a collection of natural products, disrupted important B. cenocepacia phenotypes, including motility, biofilm formation, protease production, and virulence. Genetic and biochemical analyses showed that oridonin inhibited the production of BDSF and AHL signals by decreasing the expression of their synthase-encoding genes. Furthermore, we revealed that oridonin directly binds to the regulator RqpR of the two-component system RqpSR that dominates the above-mentioned QS systems to inhibit the expression of the BDSF and AHL signal synthase-encoding genes. Oridonin also binds to the transcriptional regulator CepR of the cep AHL system to inhibit its binding to the promoter of bclACB. These findings suggest that oridonin could potentially be developed as a new QS inhibitor against pathogenic B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is an important human opportunistic pathogen that can cause life-threatening infections in susceptible individuals. It employs quorum-sensing (QS) systems to regulate biological functions and virulence. In this study, we have identified a lead compound, oridonin, that is capable of interfering with B. cenocepacia QS signaling and physiology. We demonstrate that oridonin suppressed cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) signal production and attenuated virulence in B. cenocepacia. Oridonin also impaired QS-regulated phenotypes in various Burkholderia species. These results suggest that oridonin could interfere with QS signaling in many Burkholderia species and might be developed as a new antibacterial agent.

There is an antivirulence screen with more than 1,000 natural products that could be made available Fig. 1 and 2 show several compounds that seem to have similar activity to that of oridonin. However, the choice of oridonin for follow-up studies is not justified.
Line 136-139. There is a knowledge gap between a compound's first report of activity and the identification of its putative binding target. It is not clear what knowledge or findings led the authors to hypothesize that RqpR could be a binding target of oridonin. The literature reports several effects of Oridonin seems on many celullar process. It seems that many of these claims could be due to unspecific binding, protein aggregation, protein precipitation. Have the authors considered this posibility?
In order to show activity, most of the assays are performed at high concentrations(100uM). There is plenty of data on the toxic effects of oridonin. Have the authors look at the toxic effects at these concentrations? Figure 6A and 6B. It is not clear how the authors calculated the percentages. The y-axis of figure 6B is labeled % WT. It is not clear what that means.
The idea of targeting quorum sensing as an antivirulent target is not new. However, it did not so far render any promising results. The authors should exert caution on suggesting this approach as promising. Quorum sensing in Burkholderia is complex and not conserved in the different species. In addition, quorum sensing may not be relevant at chronic stages of infection.

Reviewer #2 (Comments for the Author):
This study showed the inhibitory efficacy of oridonin against two types of quorum sensing in the pathogenic Burkholderia cenocapacia. The various experimental data support the conclusions, and the manuscript is written in standard English and easy to comprehend. However, 1. Lack of description of the rationale for choosing oridonin as a QS inhibitor among all the other candidates. 2. The concentration of 100 μM, which showed effectiveness as a QS inhibitor, is pretty much high. Is it economical for practical use? 3. There is no discussion about the mode of action of oridonin as a QS inhibitor. Since the chemical structures of oridonin, BDSF and C8-HSL are different, it is less likely that the oridonin is a competitive inhibitor. It can easily get the evidence through a simple experiment to determine whether the oridonin is a non-competitive inhibitor. Please test whether increasing the concentration of C8-HSL does not affect biofilm, motility, or protease activity in the cepI mutant in the absence or presence of oridonin at the IC50 value.
The following are minor points that are needed to improve the manuscript. 1) Lines 97, 215: "Burkholderia" should be italic. 2) Lines 111-113: data not shown? 3) Lines 118-121: The levels of cytotoxicity of bilirubin look similar to its theaflavin 3'3-digallate. Please clarify the percentage of cytotoxicity as the baseline for choosing the compounds. 4) Lines 128-133: Based on your data ( Fig. 2A), β-hydroxylsovalerylshikonin is not an effective inhibitor against rpfF gene expression. Same as the theaflavin 3'3-digallate, protopseudohypericin, ginsenoside Rk1, theaflavin-3-digallate, and α-boswellic acid. Please consider re-select the compounds which show the significant differences. 5) Lines 200-201: Please explain the reason in the Discussion. 6) Methods: If you used a commercial product of oridonin, please mention the company of the product. 7) Figure 4B and lines 173-175: The intensities of the band of the bound probe are pretty much the same between 20 μM and 10 μM of oridonin. It seems that the amount of the bclACB promoter probe bound to the CepR did not decrease dependent on increasing oridonin concentration. 8) Method: In general, QS signal receptor proteins are insoluble when overexpressed without the cognate signals. Please describe the details of the purification process of signal receptor proteins, and indicate whether the QS signals were added to the EMSA reaction.
Staff Comments:

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Comments and Suggestions for the Author:
This study showed the inhibitory efficacy of oridonin against two types of quorum sensing in the pathogenic Burkholderia cenocapacia. The various experimental data support the conclusions, and the manuscript is written in standard English and easy to comprehend. However, 1. Lack of description of the rationale for choosing oridonin as a QS inhibitor among all the other candidates.
2. The concentration of 100 μM, which showed effectiveness as a QS inhibitor, is pretty much high.
Is it economical for practical use?
3. There is no discussion about the mode of action of oridonin as a QS inhibitor. Since the chemical structures of oridonin, BDSF and C8-HSL are different, it is less likely that the oridonin is a competitive inhibitor. It can easily get the evidence through a simple experiment to determine whether the oridonin is a non-competitive inhibitor. Please test whether increasing the concentration of C8-HSL does not affect biofilm, motility, or protease activity in the cepI mutant in the absence or presence of oridonin at the IC 50 value.
The following are minor points that are needed to improve the manuscript.

Point-to-point response to reviewers' suggestions
Reviewer comments: Reviewer #1 (Comments for the Author): in this work, Li et al identify oridonin among more than 1,000 natural compounds as an antivirulence and anti-quorum sensing molecule in Burkholderia cenocepacia. They claim that oridonin could be used for treating Burkholderia infections.

Major comments
There is an antivirulence screen with more than 1,000 natural products that could be made available Fig. 1 and 2 show several compounds that seem to have similar activity to that of oridonin. However, the choice of oridonin for follow-up studies is not justified. Response: Good suggestion. Among these thirteen active candidate compounds, theaflavin-3,'3-digallate, thonningianin A, oridonin and acetyl-alpha-boswellic acid exhibited good inhibitory activity on both rpfF BC and cepI gene expression. However, we have only identified the direct targets of oridonin. Isothermal Titration Calorimetry (ITC) analysis showed that only oridonin bound to RqpR with an estimated dissociation constant (K D ) of 8.28 ± 0.895 μM (Fig.3, Fig. S2). So, we chose oridonin for further investigation in this study.
Line 136-139. There is a knowledge gap between a compound's first report of activity and the identification of its putative binding target. It is not clear what knowledge or findings led the authors to hypothesize that RqpR could be a binding target of oridonin. Response: Good suggestion. Our previous study showed that the novel two-component system RqpSR directly controls the BDSF and AHL QS systems in B.
cenocepacia (Cui et al., Molecular Microbiology, 2018). As oridonin significantly inhibited both cepI and rpfF BC gene expression (Fig.2), we then tested whether oridonin affects the expression of rpfF BC and cepI through RqpR. These details have been described in line 136-140. The literature reports several effects of Oridonin seems on many celullar process. It seems that many of these claims could be due to unspecific binding, protein aggregation, protein precipitation. Have the authors considered this posibility? Response: Good suggestion. In this study, we have identified two direct targets of oridonin, RqpR and CepR ( Fig.3 and Fig.4). However, it is possible that there are other unknown targets of oridonin, or other mechanisms employed by oridonin to affect the functions of B. cenocepacia, which needs further investigation.
In order to show activity, most of the assays are performed at high concentrations(100uM). There is plenty of data on the toxic effects of oridonin. Have the authors look at the toxic effects at these concentrations? Response: Thanks for your good suggestion. In this study, we found that oridonin could significantly reduce the production of quorum sensing signals, inhibit the biofilm formation, motility and protease activity of B. cenocepacia at a final concentration from 20-100 μM (Fig.3, Fig.4 and Fig.5). In addition, we also found that oridonin remarkably attenuated B. cenocepacia virulence, while exerted nontoxic effect towards A549 cells at a final concentration from 12.5 to 100 μM (Fig. 6). The idea of targeting quorum sensing as an antivirulent target is not new. However, it did not so far render any promising results. The authors should exert caution on suggesting this approach as promising. Quorum sensing in Burkholderia is complex and not conserved in the different species. In addition, quorum sensing may not be relevant at chronic stages of infection. Response: Good suggestion, we have revised the sentence as suggested (Page 1, Line 89). Our study just showed that oridonin inhibited QS of B. cenocepacia H111 and reduced virulence and inflammation caused by B. cenocepacia H111, but whether the quorum sensing systems play a role in the chronic infection of B. cenocepacia still needs further study.

Reviewer #2 (Comments for the Author):
This study showed the inhibitory efficacy of oridonin against two types of quorum sensing in the pathogenic Burkholderia cenocapacia. The various experimental data support the conclusions, and the manuscript is written in standard English and easy to comprehend. However, 1. Lack of description of the rationale for choosing oridonin as a QS inhibitor among all the other candidates. Response: Good suggestion. Among these thirteen active candidate compounds, theaflavin-3,'3-digallate, thonningianin A, oridonin and acetyl-alpha-boswellic acid exhibited good inhibitory activity on both rpfF BC and cepI gene expression. However, we have only identified the direct targets of oridonin. Isothermal Titration Calorimetry (ITC) analysis showed that only oridonin bound to RqpR with an estimated dissociation constant (K D ) of 8.28 ± 0.895 μM (Fig.3, Fig. S2). So, we chose oridonin for further investigation in this study.
2. The concentration of 100 μM, which showed effectiveness as a QS inhibitor, is pretty much high. Is it economical for practical use? Response: Thanks for your good suggestion. In this study, we found that oridonin could significantly reduce the production of quorum sensing signals, inhibit the biofilm formation, motility and protease activity of B. cenocepacia at a final concentration from 20-100 μM (Fig.3, Fig.4 and Fig.5). In addition, we also found that oridonin remarkably attenuated B. cenocepacia virulence, while exerted nontoxic effect towards A549 cells at a final concentration from 12.5 to 100 μM (Fig. 6).
3. There is no discussion about the mode of action of oridonin as a QS inhibitor. Since the chemical structures of oridonin, BDSF and C8-HSL are different, it is less likely that the oridonin is a competitive inhibitor. It can easily get the evidence through a simple experiment to determine whether the oridonin is a non-competitive inhibitor. Please test whether increasing the concentration of C8-HSL does not affect biofilm, motility, or protease activity in the cepI mutant in the absence or presence of oridonin at the IC50 value. Response: Good suggestion. We have added the biofilm formation experiments as suggested. Our results showed that BDSF and C8-HSL increased the biofilm formation of rpfF BC and cepI mutants, respectively, in a dose-dependent manner in the absence of oridonin (Fig.S6, S7). Exogenous addition of 50 μM BDSF and C8-HSL fully rescued the impaired biofilm formation of rpfF BC and cepI mutants, respectively, in the absence of oridonin, while exhibited no any restored effects on the biofilm formation of the signal-minus mutants in the presence of 100 μM oridonin (Fig.S6, S7). These results suggest the complicated action mechanisms and multiple targets of oridonin in B. cenocepacia, which needs further investigation. We have added these results in the revised version of our manuscript.
The following are minor points that are needed to improve the manuscript. 1) Lines 97, 215: "Burkholderia" should be italic. Response: We have revised it as suggested.
2) Lines 111-113: data not shown? Response: We have revised it as suggested.
3) Lines 118-121: The levels of cytotoxicity of bilirubin look similar to its theaflavin 3'3-digallate. Please clarify the percentage of cytotoxicity as the baseline for choosing the compounds. Response: Thanks for your good suggestion. We have modified this sentence. 4) Lines 128-133: Based on your data ( Fig. 2A), β-hydroxylsovalerylshikonin is not an effective inhibitor against rpfF gene expression. Same as the theaflavin 3'3-digallate, protopseudohypericin, ginsenoside Rk1, theaflavin-3-digallate, and α-boswellic acid. Please consider re-select the compounds which show the significant differences. Response: Thanks for your good suggestion, we have revised this sentence as suggested. 5) Lines 200-201: Please explain the reason in the Discussion. Response: Thanks for your good suggestion, we have revised it as suggested. 6) Methods: If you used a commercial product of oridonin, please mention the company of the product. Response: Thanks for your good suggestion. We have added more information of oridonin in the Methods section as suggested. 7) Figure 4B and lines 173-175: The intensities of the band of the bound probe are pretty much the same between 20 μM and 10 μM of oridonin. It seems that the amount of the bclACB promoter probe bound to the CepR did not decrease dependent on increasing oridonin concentration. Response: Good suggestion, we have repeated the EMSA experiments and replaced the picture. 8) Method: In general, QS signal receptor proteins are insoluble when overexpressed without the cognate signals. Please describe the details of the purification process of signal receptor proteins, and indicate whether the QS signals were added to the EMSA reaction. Response: Good suggestion, we have added more information in the Methods section as suggested. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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